PLEASE READ!!
HELENA LABORATORIES
PROCEDURE DOWNLOAD END USER AGREEMENT
In response to customer requests, Helena is pleased to provide the text for procedural package inserts in a digital
format editable for your use. The text for the procedure you requested begins on page three of this document.
Helena procedures contain the content outlined in the NCCLS (GP2-A#) format, except in the order sequence
required by FDA regulations. As the NCCLS format is a guideline, you may retain these procedures as developed
by the manufacturer (adding your title/authorization page) or manipulate the text file to produce your own document,
matching the NCCLS section order exactly, if preferred.
We also provide the procedure in an Adobe Acrobat PDF format for download at www.helena.com as a “MASTER”
file copy. Below are the specifications and requirements for using these digital files. Following the specifications is
the procedure major heading sequence as given in the FDA style. Where there is a difference in order, or other
notation in the outline, this will be indicated in braces { }.
WHAT YOU NEED TO KNOW:
1)
These files represent the most current revision level to date. Your current product inventory could contain
a previous revision level of this procedure.
2)
The Microsoft Word document provides the text only from the master procedure, in a single-column format.
-
It may not contain any illustrations, graphics or captions that may be part of the master procedure
included in the kit.
-
The master procedure may have contained special formatting characters, such as subscripts,
superscripts, degree symbols, mean symbols and Greek characters such as alpha, beta, gamma, etc.
These symbols may or may not display properly on your desktop.
-
The master procedures may also contain columns of tabbed data. Tab settings may or may not be
displayed properly on your desktop.
3)
The Adobe Acrobat PDF file provides a snapshot of the master procedure in a printable 8.5 x 11” format. It is
provided to serve as a reference for accuracy.
4)
By downloading this procedure, your institution is assuming responsibility for modification and usage.
HELENA LABORATORIES
PROCEDURE DOWNLOAD END USER AGREEMENT
HELENA LABORATORIES LABELING – Style/Format Outline
1)
2)
3)
4)
PRODUCT {Test} NAME
INTENDED USE and TEST TYPE (qualitative or qualitative)
SUMMARY AND EXPLANATION
PRINCIPLES OF THE PROCEDURE
{NCCLS lists SAMPLE COLLECTION/HANDLING next}
5)
REAGENTS (name/concentration; warnings/precautions; preparation; storage; environment;
Purification/treatment; indications of instability)
6)
INSTRUMENTS required – Refer to Operator Manual (... for equipment for; use or function; Installation;
Principles of operation; performance; Operating Instructions; Calibration* {*is next in order for NCCLS – also
listed in “PROCEDURE”}’ precautions/limitations/hazards; Service and maintenance information
7)
SAMPLE COLLECTION/HANDLING
8)
PROCEDURE
{NCCLS lists QUALITY CONTROL (QC) next}
9) RESULTS (calculations, as applicable; etc.)
10) LIMITATIONS/NOTES/INTERFERENCES
11) EXPECTED VALUES
12) PERFORMANCE CHARACTERISTCS
13) BIBLIOGRAPHY (of pertinent references)
14) NAME AND PLACE OF BUSINESS OF MANUFACTURER
15) DATE OF ISSUANCE OF LABELING (instructions)
For Sales, Technical and Order Information, and Service Assistance,
call Helena Laboratories toll free at 1-800-231-5663.
Form 364
Helena Laboratories
1/2006 (Rev 3)
Thromboplastin LI
Prothrombin Time Reagent
INTENDED USE
Thromboplastin LI is intended for use in a one-stage prothrombin time (PT) test on citrated human plasma. The PT test is a quantitative
assay used in the general patient population for routine screening to detect deficiencies in the extrinsic pathway of coagulation. The PT
test is also used to monitor oral anticoagulant therapy and should be used in a clinical laboratory by qualified laboratory personnel.
SUMMARY AND PRINCIPLE
The one-stage prothrombin time test (PT) has become the basic coagulation screening test for the diagnosis of congenital and acquired
1, 2
deficiencies of Factors II, V, VII and X . Oral anticoagulant drugs inhibit hepatic synthesis of the vitamin K dependent clotting Factors II,
VII, IX and X. Therefore, the prothrombin time test is appropriate to monitor oral anticoagulant therapy since it is sensitive to three of the
3, 4
four factors involved . It can be modified to quantitate factor levels using appropriate factor deficient substrate plasmas. Tissue
thromboplastin, in the presence of calcium ions and Factor VII, activates the extrinsic pathway of coagulation. When a mixture of tissue
thromboplastin and calcium ions is added to normal anti-coagulated plasma, the clotting mechanism is initiated and a clot will form within
a specified time period. If a deficiency exists within the extrinsic pathway, the time required for clot formation will be prolonged. The
degree of prolongation is proportional to the severity of single factor deficiency, or in a cumulative deficiency of all the factors involved.
REAGENTS
1. Thromboplastin LI Reagent Ingredients: The reagent contains a liquid saline extract of rabbit brain, calcium ions, preservatives
and stabilizers.
WARNING: FOR IN-VITRO DIAGNOSTIC USE ONLY. Preparation for Use: Reagent comes ready for use. Invert vial
3-4 times before use to ensure homogeneity. Storage and Stability: Reagent should be stored at 2 to 8°C and is stable until the
expiration date indicated on the vial. After opening, the original activity is stable for fourteen (14) days at 2 to 8°C. DO NOT FREEZE.
Signs of Deterioration: The reagent is a fine suspension of rabbit brain particles. Large flaky particles in the suspension or
prolonged prothrombin times on testing normal plasma or controls may indicate product deterioration.
INSTRUMENTS
Prothrombin time test may be done by accepted manual methods or by using mechanical or photo-optical coagulation instruments such
as the Cascade M and Cascade M-4.
SPECIMEN COLLECTION AND HANDLING NOTE: After initial whole blood collection with 3.2% sodium citrate, all test
tubes, syringes and pipettes used during testing should be plastic. Specimen: Plasma obtained from whole blood anti-coagulated
with 0.1 M (3.2%) sodium citrate. Specimen Collection: Nine parts freshly collected whole blood should be immediately added to one
part anticoagulant.
Specimen Preparation: Specimens may be stored uncentrifuged or centrifuged with plasma remaining on the cells in an unopened
tube at 18 to 24°C (CLSI H21-4, 2003). To obtain a plasma sample, the capped specimen tube should be centrifuged at 1500 x g for 15
minutes and the plasma removed with a plastic pipette, placed in a plastic tube and capped. PT assays should be completed within 24
hours of collection. If testing is not completed within 24 hours, plasma should be removed from cells and frozen at -20°C for up to 2
weeks or -70°C for up to 6 months.
Storage and Stability: Before and during testing, the plasma sample should be maintained in the plastic tubes at 2 to 8°C to ensure
stability of the factors. If testing is delayed for more than 2 hours, the plasma may be stored at -20°C for two weeks or at -70°C for up to
one month. Frozen samples should be thawed rapidly at 37°C before testing.
PROCEDURE Materials Provided: Material needed for prothrombin time tests are provided in the following packaging configurations:
Cat. No.
Thromboplastin LI Reagent (10 x 5 mL vials)
5248
Thromboplastin LI Reagent (10 x 10 mL vials)
5249
Materials and Equipment Required, but not provided:
1. Coagulation Instrument or 37°C Water Bath and timer
2. Reaction Cups or plastic test tubes (12 x 75 mm)
3. Pipetting device to deliver 200 µL or 100 µL
4. Centrifuge
5. Distilled or deionized water
6. Control plasmas: Norm-Trol 1 – Cat. No. 5186 Ab-Trol 2 - Cat. No. 5187 Ab-Trol 3 - Cat. No. 5183
PROCEDURE NOTE: Throughout the procedure, all test tubes, syringes, and pipettes should be plastic.
Automated and Semi-Automated Methods
If using an instrument to perform this test, refer to the appropriate Instrument Operator’s Manual for detailed instructions.
Quality Control:
Each laboratory should establish a quality control program that includes normal and abnormal controls to evaluate instrument, reagent
and technologist performance. The normal and abnormal controls should be tested daily prior to performing tests on patient plasmas.
Monthly quality control charts (Levy Jennings) are recommended to determine the mean and standard deviation of each of the daily
control plasmas. A normal control such as Norm-Trol 1, and abnormal level 1 and level 2 controls such as Ab-Trol 2 and Ab-Trol 3 are
recommended. If the controls do not perform within their reference ranges, patient results should be considered invalid and not reported.
EXPECTED VALUES
A reference range study was conducted using triplicate specimens from 120 normal healthy adults. Approximately equal numbers of
males and females were used. The PT results were as follows:
Photo-optical
Mechanical
Mean (seconds)
12.3
12.6
Range + 2 SD
9.7 - 14.9 seconds
11.1 - 14.1 seconds
These values should only serve as guidelines. Because differences may exist between instruments, laboratories, and local populations,
it is recommended that each laboratory establish its own reference range of expected prothrombin time results.
DETERMINATION OF INR
The World Health Organization (WHO) recommends the use of the International Normalized Ratio (INR) instead of direct reporting of the
PT times when monitoring patients undergoing Oral Anticoagulant Therapy. This allows patient results to be compared between different
laboratories that may be using reagents with different sensitivities.
The INR is calculated by using the ratio of the Patient PT to the mean of the normal reference range raised to the power of the reagent
International Sensitivity Index (ISI).
ISI
INR = (Patient PT/mean of normal range)
The ISI is assigned by comparison to a highly sensitive WHO thromboplastin standard reference material. The lower the ISI for
a given reagent the more sensitive the reagent is to coagulation factors. The lot specific ISI value for Thromboplastin LI can be
found on the enclosed ISI calculation sheet.
RESULTS
The results of the Prothrombin Time tests should be reported to the nearest tenth of a second. Results greater than the upper limits of
the range should be considered abnormal and follow-up testing should be performed. PT values below the lower limits of the range may
indicate a compromised sample, and a new sample should be collected.
LIMITATIONS
Expected values for the prothrombin time test will vary from one laboratory to another, depending on several variables. These include
the method of clot detection, temperature, pH, sample collection technique, type of anticoagulant and time and method of plas ma
storage. Therefore, laboratories should establish their own expected values for patients and well defined performance standards for
control plasmas. The use of icteric, lipemic, or hemolyzed sample should be avoided due to possible interference especially when using
photo-optical instruments. The impact of other therapeutic drugs, in addition to oral anticoagulant therapy, can influence interpretation of
PT test results. Obtaining an accurate patient history and noting specific drug therapies can help in the proper understanding of the
potential impact on laboratory test results. The presence of heparin as a contaminant in the patient sample must always be considered
when an abnormal result is obtained.
PERFORMANCE CHARACTERISTICS PRECISION
Precision studies were performed to establish Within-Run and Between-Run CV’s for normal control plasma and abnormal control
plasma. A single lot number of Thromboplastin LI reagent was used for these studies. Results are shown below
Photo-optical
Norm-Trol
Ab-Trol 2
Ab-Trol 3
Within Run
Between Run
n
Mean
20
13.7
60
13.7
SD
CV%
0.2
1.7
0.2
1.3
n
Mean
20
31.3
60
31.1
SD
CV%
0.5
1.6
0.6
1.8
Mean
20
66.6
60
68.6
SD
1.5
2.1
CV%
2.2
3.1
n
Mechanical
Norm-Trol
n
Mean
SD
CV%
Within Run
20
14.1
0.3
2.0
Between Run 20 60
60
14.4
0.36
2.5
Ab-Trol 2
n
Mean
SD
CV%
20
32.9
0.9
2.8
60
2.4
1.1
3.4
Ab-Trol 3
n
Mean
SD
CV%
20
70.4
2.7
3.9
60
73.5
3.4
4.7
COMPARISON
A comparison study was done using the Thromboplastin LI Reagent and another thromboplastin reagent. One hundred five (105)
plasma specimens, both normal and abnormal clinical samples were tested with both PT reagents. The linear regression equation and
coefficient of determination (r ) of the INR values are reported.
2
Photo-optical
N = 105
Mechanical
N = 105
r2 = 0.98
Y = 1.065X – 0.1245
r2 = 0.92
Y = 1.059X – 0.114
Y = Thromboplastin LI reagent
X = Reference thromboplastin reagent
References:
1. Quick A.J., The Prothrombin Time in Hemophilia and in Obstructive Jaundice. J. Biol. Chem:109,73-74;1935.
2. Biggs R. ed, Human Blood Coagulation Hemostasis and Thrombosis Second Ed. Blackwell Scientific Publications, London 1976.
3. Peterson C.E., Kwaan H.C., Current Concepts of Warfarin Therapy, Arch Intern. Med. 146:581-584, 1986.
4. Loeliger E.A:ICEH/ICTH Recommendations for Reporting Prothrombin Time in Oral Anticoagulant Control, Throm. Haemost.
53:155-156, 1985.
THROMBOPLASTIN LI Reagent
Thromboplastin LI Reagent (10 x 5 mL)
Thromboplastin LI Reagent (10 x 10 mL)
Cat. No.
5248
5249
EQUIPMENT AND SUPPLIES
Coagulation S.A.R.P. (10 x 1.0 mL)
Norm-Trol Coagulation Control (10 x 1.0 mL)
Ab-Trol 1 Coagulation Control (10 x 1.0 mL)
Ab-Trol 2 Coagulation Control (10 x 1.0 mL)
Cascade M
Cascade M-4
5185
5186
5187
5183
1710
1711
For Sales, Technical and Order Information and Service Assistance, call 800-231-5663 toll free.
Helena Laboratories warrants its products to meet our published specifications and to be free from defects in materials and workmanship. Helena’s liability under this
contract or otherwise shall be limited to replacement or refund of any amount not to exceed the purchase price attributable to the goods as to which such claim is
made. These alternatives shall be buyer’s exclusive remedies. In no case will Helena Laboratories be liable for consequential damages even if Helena has been
advised as to the possibility of such damages. The foregoing warranties are in lieu of all warranties expressed or implied including, but not limited to, the implied
warranties of merchantability and fitness
for a particular purpose.
European Community Authorized Representative Helena Laboratories UK, Ltd. Colima Avenue Sunderland Enterprise
Park Sunderland SR5 3XB England
Pro. 121
3/07(3)